Tethered detection assays

Abstract

Disclosed herein, inter alia, are compositions, complexes, and methods useful for efficient multiplex assays.

What is claimed is: 
     
         1 . A method of detecting a biomolecule in a sample, said method comprising:
 contacting a solid support with a sample comprising a biomolecule, wherein said biomolecule is a lipid, carbohydrate, peptide, protein, or antigen binding fragment, and said solid support comprises a first biomolecule-specific binding agent attached to the solid support; and a second biomolecule-specific binding agent attached to said solid support via a cleavable linker, thereby forming a complex comprising the first biomolecule-specific binding agent bound to the biomolecule and the second biomolecule-specific binding agent bound to the biomolecule;   cleaving the cleavable linker of the complex thereby forming a cleaved complex;   wherein cleaving comprises contacting the cleavable linker with a cleaving agent and detaching the second biomolecule-specific binding agent from the solid support;   detecting the solid support; and detecting the cleaved complex.   
     
     
         2 . The method of  claim 1 , wherein the second biomolecule-specific binding agent comprises a detectable moiety. 
     
     
         3 . The method of  claim 1 , wherein the cleavable linker comprises a polynucleotide or a polypeptide sequence. 
     
     
         4 . The method of  claim 1 , wherein the cleavable linker comprises a polynucleotide sequence. 
     
     
         5 . The method of  claim 1 , wherein cleaving the cleavable linker comprises generating an identification oligonucleotide, wherein said identification oligonucleotide comprises a portion of the polynucleotide sequence. 
     
     
         6 . The method of  claim 1 , further comprising amplifying the identification oligonucleotide to generate amplification products. 
     
     
         7 . The method of  claim 6 , wherein detecting the biomolecule comprises detecting the identification oligonucleotide or one or more of the amplification products. 
     
     
         8 . The method of  claim 2 , wherein detecting the biomolecule comprises detecting the detectable moiety. 
     
     
         9 . The method of  claim 1 , wherein the second biomolecule-specific binding agent further comprises a bioconjugate reactive moiety, an enzyme, or a label. 
     
     
         10 . The method of  claim 1 , wherein the cleaving agent comprises a reducing agent, sodium periodate, or a nuclease. 
     
     
         11 . The method of  claim 1 , wherein the cleaving agent comprises cleaving agent comprises RNase, Formamidopyrimidine DNA Glycosylase (Fpg), a restriction enzyme, or uracil DNA glycosylase (UDG). 
     
     
         12 . The method of  claim 1 , wherein the first biomolecule-specific binding agent and the second biomolecule-specific binding agent are each independently an antibody, single-chain Fv fragment (scFv), antibody fragment-antigen binding (Fab), affimer, or an aptamer. 
     
     
         13 . The method of  claim 1 , wherein the first biomolecule-specific binding agent and the second biomolecule-specific binding agent are each an antibody. 
     
     
         14 . The method of  claim 1 , wherein the first biomolecule-specific binding agent is a monoclonal antibody and the second biomolecule-specific binding agent is a polyclonal antibody. 
     
     
         15 . The method of  claim 1 , wherein the solid support is a particle comprising two or more fluorescent dyes. 
     
     
         16 . The method of  claim 15 , wherein the two or more fluorescent dyes are selected from cyclobutenedione derivatives, symmetrical and unsymmetrical squaraines, substituted cephalosporin compounds, fluorinated squaraine compositions, alkylalkoxy squaraines, or squarylium compounds. 
     
     
         17 . The method of  claim 16 , wherein the two or more fluorescent dyes are selected from 1,3-bis[(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)methyl]-2,4-dihydroxycyclobutenediylium, bis(inner salt) and 2-(3,5-dimethylpyrrol-2-yl)-4-(3,5-dimethyl-2H-pyrrol-2-ylidene)-3-hydroxy-2-cyclobuten-1-one. 
     
     
         18 . The method of  claim 1 , wherein the solid support is a particle comprising polystyrene, brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyacrylamide, polyacrolein, polydimethylsiloxane, polybutadiene, polyisoprene, polyurethane, polyvinyl acetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivindylbenzene, polyglycidylmethacrylate, polymethylmethacrylate, or copolymers, blends, composites, or combination thereof. 
     
     
         19 . The method of  claim 1 , wherein detecting the solid support comprises detecting the two or more fluorescent dyes. 
     
     
         20 . The method of  claim 1 , wherein the solid support comprises a substrate polynucleotide barcode, or the first biomolecule-specific binding agent comprises a substrate polynucleotide barcode. 
     
     
         21 . The method of  claim 20 , wherein detecting the solid support comprises detecting substrate barcode. 
     
     
         22 . A solid support, comprising:
 a first biomolecule-specific binding agent attached to the solid support;   a second biomolecule-specific binding agent attached to the solid support via a cleavable linker, wherein the cleavable linker is a divalent linker comprising one or more cleavable sites, wherein the second biomolecule-specific binding agent comprises a detectable moiety.   
     
     
         23 . The solid support of  claim 22 , wherein the cleavable linker is a covalent cleavable linker. 
     
     
         24 . The solid support of  claim 22 , further comprising one or more oligonucleotides, wherein the one or more oligonucleotides are attached to the solid support. 
     
     
         25 . The solid support of  claim 22 , wherein the solid support is a particle. 
     
     
         26 . The solid support of  claim 25 , wherein said particle is in a well of a multiwell container. 
     
     
         27 . The solid support of  claim 25 , wherein the average longest dimension of said particle is from about 100 nm to about 3000 nm. 
     
     
         28 . The solid support of  claim 25 , wherein said particle comprises glass, ceramic, metal, silica, magnetic material, or a paramagnetic material. 
     
     
         29 . A plurality of particles, wherein each of said particles is independently a particle of  claim 25 . 
     
     
         30 . A method of amplifying a polynucleotide sequence attached to a biomolecule-specific binding agent, said method comprising:
 contacting a solid support with a biomolecule, wherein said solid support comprises:
 a first biomolecule-specific binding agent attached to the solid support; 
 a second biomolecule-specific binding agent attached to the solid support via a cleavable linker, wherein said cleavable linker comprises the polynucleotide sequence; 
   forming a complex comprising a biomolecule bound to both the first biomolecule-specific binding agent bound and the second specific-binding agent;   cleaving the cleavable linker of the complex thereby forming a cleaved complex; wherein cleaving comprises contacting the cleavable linker with a cleaving agent and detaching the second biomolecule-specific binding agent from the solid support; and   hybridizing a primer oligonucleotide to the polynucleotide sequence and extending the primer oligonucleotide sequence with a polymerase, thereby amplifying the polynucleotide sequence attached to the second biomolecule-specific binding agent.   
     
     
         31 . The method of  claim 30 , wherein the cleavable linker is a covalent cleavable linker.